Heterotrimeric G proteins are eminent signal transducing molecules that facilitate human sensation and hormone-regulation of physiology. Traditional G protein signaling pathways are activated by the action of G protein coupled receptors (GPCRs) present on the surface of cells. Recently, new mechanisms of heterotrimeric G protein regulation have been appreciated. Enzymes that are not GPCRs activate G proteins by non-traditional means and regulate pathways not previously attributed to G protein control. Prototypes of these new G protein activators are the Ric-8 proteins. The mammalian Ric-8A homolog activates G protein alpha subunits by stimulating catalysis of GDP for GTP exchange. Ric-8 proteins likely modulate G protein signaling regulation of asymmetric (adult stem cell) and normal cell division (G protein alpha i), aspects of neurotransmission (G protein alpha q), and olfaction (G protein alpha olf). Perhaps underlying this seeming regulation of divergent G protein signaling pathways is the finding that Drosophila RIC-8 appears to be required for the proper expression and plasma membrane localization of heterotrimeric G proteins. The goal of this proposal is to understand in detail, the unique regulation of G protein physiology by both mammalian Ric-8 homologs (A and B). The aims of the proposal are to: (1) examine the hypothesis that Ric-8B proteins activate G protein alpha s subunits by serving as guanine nucleotide exchange catalysts, (2) test the hypothesis in mammals, that Ric-8 proteins are required globally for G protein localization at the cell plasma membrane. (3) test the hypothesis that the mammalian Ric-8A homolog is responsible for activating G protein alpha i- dependent (asymmetric) cell division (4) define the authentic cellular interactions between the compendium of G protein subunits and Ric-8A or Ric-8B. The technology developed to define these interactions will also be used to uncover novel interactions between Ric-8 proteins and other proteins in addition to G proteins. Ric-8 proteins are new G protein activators. Many pharmaceuticals intervene to alter the process of GPCR-mediated G protein activation. Elucidation of the novel mechanisms by which Ric-8 proteins regulate G proteins may enable the engineering of new classes of therapeutics that intervene at the level of Ric-8 activation of G proteins. Furthermore, contributing basic knowledge towards understanding the mechanism(s) by which Ric-8 and G proteins control normal and defective stem cell division could profit: (1) the therapeutic use of stem cells to repair damaged tissues and (2) treatment of stem cell derived cancers. PUBLIC HEALTH RELEVANCE: Heterotrimeric G protein activation is a primary target of pharmaceutical intervention. Defining the unique mechanisms by which Ric-8 proteins activate G proteins will enable the design of new classes of therapeutics that alter the consequences of G protein activation. This research will also define Ric-8 control of G protein-directed stem cell division and contribute efforts to use stem cells for tissue repair and the eradication of cancers derived from defectively dividing stem cells.